TW202321297A - Anti-CD47-CLDN18.2 bispecific antibody and use thereof - Google Patents

Abstract

The present invention relates to the field of biological medicines. Specifically, the present invention relates to an anti-CD47-CLDN18.2 bispecific antibody and use thereof.

Description

Anti-CD47-CLDN18.2 bispecific antibodies and their uses

The present invention belongs to the field of biomedicine. Specifically, the present invention relates to anti-CD47-CLDN18.2 bispecific antibodies and their uses.

CD47 is a transmembrane glycoprotein widely expressed on the cell surface. It belongs to the immunoglobulin superfamily and can interact with signal regulatory protein α (SIRPα), platelet activin (Thrombospondin-1, TSP1) and integrin (Integrin). ) interact to mediate a series of reactions such as cell apoptosis, proliferation, and immunity. It has been confirmed that blocking the CD47-SIRPα pathway through the use of anti-CD47 antibodies can effectively mediate phagocytosis of tumor cells, thereby inhibiting the growth of various hematological tumors and solid tumors in vivo. However, CD47 is highly expressed not only on tumor cells, but also on normal cells, such as red blood cells. Therapies targeting CD47 may cause undesirable side effects. Some anti-CD47 antibodies disclosed in the prior art (see, for example, US20160304609) bind to red blood cells, which not only triggers a severe anemia reaction, but also requires a dosage of up to 30 mg/kg for administration. These properties limit the clinical application of anti-CD47 antibodies. brought great challenges.

CLDN18 belongs to the Claudins protein family and was discovered by Shoichiro Tsukita et al. in 1998. It is an important molecule that constitutes the tight junction of epithelial cells, determines the permeability of epithelial cells, and also plays a role in blocking the diffusion of proteins and lipids on the cell membrane surface (Gunzel, D. and A. S. Yu (2013) Physiol Rev 932: 525-569). The human CLDN18 gene has two different exons 1, which undergo alternative splicing after transcription to eventually generate two protein isoforms CLDN18.1 and CLDN18.2 with different sequences only at the N terminus. Due to its specific expression in tumor cells and normal tissues, CLDN18.2 has become a very potential anti-tumor drug target. WO2016165762A1 discloses the anti-CLDN18.2 antibody IMAB362 (Zolbetuximab), which showed a significantly longer survival than standard chemotherapy in a phase II gastric cancer clinical trial (13.2 vs. 8.4 months), and the advantage was more obvious in patients with high CLDN18.2 expression.

WO2021003082A1 discloses anti-CLDN18.2 and CD47 bispecific antibodies that essentially do not bind human red blood cells. However, there is still a need to develop new bispecific antibodies targeting CLDN18.2 and CD47 to provide more possibilities for cancer treatment.

In one aspect, the invention provides a bispecific antibody comprising a first antigen-binding portion that binds CD47 and a second antigen-binding portion that binds CLDN18.2, wherein the first antigen-binding portion comprises a heavy chain variable region ( VH) and a light chain variable region (VL), the heavy chain variable region includes: 1) HCDR1, which includes the amino acid sequence of SEQ ID NO:4; 2) HCDR2, which includes the amino acid sequence of SEQ ID NO:5 ; and 3) HCDR3, which includes the amino acid sequence of SEQ ID NO:6; and, the light chain variable region includes: 1) LCDR1, which includes the amino acid sequence of SEQ ID NO:7; 2) LCDR2, which includes SEQ The amino acid sequence of ID NO:8; and 3) LCDR3 comprising the amino acid sequence of SEQ ID NO:9.

In one embodiment, the second antigen binding portion comprises an immunoglobulin single variable domain (VHH) that binds CLDN18.2. Preferably, the immunoglobulin single variable domain includes: CDR1, which includes the amino acid sequence of SEQ ID NO: 13; CDR2, which includes the amino acid sequence of SEQ ID NO: 14; and CDR3, which includes SEQ ID NO: 15 amino acid sequences.

In yet another aspect, the invention provides an isolated polynucleotide encoding a bispecific antibody of the invention.

The present invention also provides an expression vector comprising the polynucleotide of the present invention.

In another aspect, the invention provides a host cell comprising a polynucleotide or expression vector of the invention.

The invention also relates to antibody conjugates comprising a bispecific antibody of the invention conjugated to at least one therapeutic agent.

In yet another aspect, the invention relates to a pharmaceutical composition comprising the bispecific antibody or antibody conjugate of the invention, and a pharmaceutically acceptable carrier.

The invention also relates to the use of the bispecific antibody, antibody conjugate or pharmaceutical composition of the invention for the preparation of a medicament for the treatment of cancer.

[ definition ]

In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used in this article are terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

As used herein, the expressions "includes," "includes," "contains," and "having" are open-ended and mean the inclusion of recited elements, steps or components but not the exclusion of other unrecited elements, steps or components. The expression "consisting of" does not include any element, step or component not specified. The expression "consisting essentially of" means that the scope is limited to the specified elements, steps or components plus the presence of optional elements, steps or components that do not materially affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of" are encompassed within the meaning of the expression "including".

As used herein, "antibody" refers to an immunoglobulin or fragment thereof that specifically binds to an antigenic epitope through at least one antigen-binding site. As used herein, the definition of antibody encompasses antigen-binding fragments. The term "antibody" includes multispecific antibodies (eg, bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies, and antigen-binding fragments. Antibodies may be synthetic (eg, produced through chemical or biological conjugation), enzymatically processed, or recombinantly produced. Antibodies provided herein include any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass (e.g., IgG2a and IgG2b). Antibodies can be "monovalent," "bivalent," "trivalent," or "tetravalent," or more, meaning they contain 1, 2, 3, 4 or more antigen-binding sites.

As used herein, a "full-length antibody" generally contains four polypeptides: two heavy chains (HC) and two light chains (LC). Each light chain contains a "light chain variable region (VL)" and a "light chain constant region (CL)" from the N-terminus (amino acid end) to the C-terminus (carboxyl end). Each heavy chain includes a "heavy chain variable region (VH)" and a "heavy chain constant region (CH)" from the N-terminus to the C-terminus. Generally speaking, the heavy chain constant region of a full-length antibody may include CH1-hinge-CH2-CH3 from the N-terminus to the C-terminus. In some immunoglobulin types (such as IgM and IgE), the heavy chain constant region can contain CH1-hinge region-CH2-CH3-CH4 from N-terminus to C-terminus.

The light chain variable region and the heavy chain variable region can each contain three highly variable "complementarity determining regions (CDR)" and four relatively conserved "framework regions (FR)", and from the N-terminus to the C-terminus are The sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 is connected. In this article, the CDRs of the light chain variable region (CDRL or LCDR) may be referred to as LCDR1, LCDR2, and LCDR3, and the CDRs of the heavy chain variable region (CDRH or HCDR) may be referred to as HCDR1, HCDR2, and HCDR3.

In the present invention, the amino acid sequences of the CDRs are all shown in accordance with the AbM definition rules (the patent scope of the present invention is also a sequence shown in accordance with the AbM definition rules). However, it is well known in the art that the CDRs of an antibody can be defined in a variety of ways, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (see, for example, Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)), Kabat based on antibody sequence variability (see e.g. Kabat, E.A. et al . (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), AbM (Martin, A.C.R. and J. Allen (2007) "Bioinformatics tools for antibody engineering, ” in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH Verlag, pp. 95–118), Contact (MacCallum, R. M. et al., (1996) J. Mol. Biol. 262:732 -745), IMGT (Lefranc, M.-P., 2011 (6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, M.-P. et al., Dev. Comp. Immunol. , 27, 55-77 (2003)), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass those described above as described through the present invention. Complementary determination zone defined by any of the known schemes. Although the scope of protection claimed in the patent application scope of the present invention is the sequence shown based on the AbM definition rules, the amino acid sequences corresponding to other CDR definition rules should also fall within the protection scope of the present invention.

Therefore, when it comes to defining an antibody with a specific CDR sequence as defined in the present invention, the scope of said antibody also encompasses antibodies whose variable region sequences comprise said specific CDR sequence but which differ due to the application of different protocols (e.g. Different assignment system rules or combinations) cause the claimed CDR boundaries to be different from the specific CDR boundaries defined in the present invention.

As used herein, the terms "framework area" and "framework area" are used interchangeably. As used herein, the terms "framework region", "framework region" or "FR" residues refer to those amino acid residues in an antibody variable region other than the CDR sequences as defined above.

As used herein, "single domain antibody (sdAb)" or "nanobody" refers to an antibody that contains a single immunoglobulin variable domain (single variable domain) as a functional antigen-binding fragment. Similar to the variable regions of full-length antibodies, single variable domains typically contain CDR1, CDR2, and CDR3 that form the antigen-binding site and a supporting framework region. The single variable domain may be, for example, a variable domain of heavy-chain antibody (VHH), a shark IgNAR variable domain, a human light chain antibody variable domain, and a heavy chain antibody variable domain. domain.

As used herein, "antibody-dependent cell-mediated phagocytosis" or "ADCP" refers to a cell-mediated process in which non-specific cytotoxic cells (e.g., monocytes, Macrophages, neutrophils, and dendritic cells) recognize antibodies that bind to target cells (eg, tumor cells) and subsequently act as effector cells to phagocytose the target cells (eg, tumor cells). In some embodiments, the anti-CD47-CLDN18.2 bispecific antibodies of the invention mediate ADCP against cancer cells expressing CLDN18.2, particularly expressing CD47 and CLDN18.2.

As used herein, "percent (%) sequence identity" and "sequence identity" of amino acid sequences have art-accepted definitions, which refer to two determined by sequence alignment (such as through manual inspection or publicly known algorithms). The percentage of identity between peptide sequences. This can be determined using methods known to those skilled in the art, for example using publicly available computer software such as BLAST, BLAST-2, Clustal Omega and FASTA software.

As used herein, an amino acid sequence "derived from" or "derived from" a reference amino acid sequence is identical or homologous to part or all of the reference amino acid sequence. For example, the amino acid sequence derived from a heavy chain constant region of a human immunoglobulin may be at least 80%, at least 85%, at least 90%, at least 91% identical to the wild-type sequence of the human immunoglobulin heavy chain constant region from which it is derived. , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity.

Non-critical regions in a polypeptide (such as the CDR region of an antibody, non-critical amino acids in the framework region, amino acids in the constant region) can be modified, such as substitution, addition and/or deletion of one or more amino acids without changing the function of the polypeptide. . Those skilled in the art will understand that amino acids in non-critical regions of a polypeptide can be substituted with appropriate conservative amino acids without generally changing its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin /Cummings Pub. co., p. 224). Suitable conservative substitutions are well known to those skilled in the art. In some cases, amino acid substitutions are non-conservative substitutions. Those skilled in the art will understand that amino acid mutations or modifications can be made to antibodies or antibody fragments to change their properties, such as changing the type of glycosylation modification of the antibody, changing the ability to form interchain disulfide bonds, or changing the properties of the antibody conjugate. Preparation provides active groups. Antibodies or antigen-binding fragments thereof comprising such amino acid mutations or modifications are also encompassed within the scope of the bispecific antibodies of the invention.

The anti-CD47-CLDN18.2 bispecific antibodies of the invention or polynucleotides encoding the same can be isolated. As used herein, the expression "isolated" means that a substance (eg, a polynucleotide or a polypeptide) is separated from the source or environment in which it exists, that is, does not contain substantially any other components.

As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to an oligomer or polymer containing at least two linked nucleotides or nucleotide derivatives, which may typically include deoxyribonucleic acid (DNA ) and ribonucleic acid (RNA).

As used herein, a "vector" is a vehicle used to introduce an exogenous polynucleotide into a host cell. When the vector is transformed into an appropriate host cell, the exogenous polynucleotide is amplified or expressed. Vectors usually remain episomal, but can be designed to integrate the gene or part thereof into the chromosome of the genome. As used herein, the definition of vector encompasses plastids, linearized plastids, viral vectors, myxoids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), among others. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated virus vectors, herpes virus vectors, poxvirus vectors, and baculovirus vectors.

As used herein, the term "expression" refers to the production of RNA and/or polypeptides.

As used herein, "expression vector" refers to a vector capable of expressing a polynucleotide of interest, including DNA and RNA. For example, in an expression vector, a polynucleotide sequence (including DNA and RNA) encoding a polypeptide of interest can be operably linked to regulatory sequences (such as promoters and ribosome binding sites) that can affect the expression of the polynucleotide sequence. . Regulatory sequences may include promoter and terminator sequences, and optionally may include origins of replication, selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors may be plasmids, phage vectors, recombinant viruses, or other vectors that, when introduced into an appropriate host cell, result in expression of the polynucleotide of interest. Suitable expression vectors are well known to those skilled in the art. Those skilled in the art can prepare the expression vector into a vector that can replicate in the host cell, remain free in the host cell, or be integrated into the genome of the host cell as needed.

As used herein, a "host cell" is a cell used to receive, maintain, replicate or amplify a vector. Host cells can also be used to express polypeptides encoded by polynucleotides or vectors. The host cell can be a eukaryotic cell or a prokaryotic cell. Prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells (such as S2 Drosophila cells or Sf9), and animal cells (such as fibroblasts, CHO cells , COS cells, HeLa cells, NSO cells or HEK293 cells).

As used herein, the term "treatment" refers to the improvement of a disease/symptom, such as reducing or eliminating the disease/symptom, preventing or slowing the occurrence, progression and/or worsening of the disease/symptom. Treatment therefore includes prevention, treatment and/or cure.

As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient and is well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents Reagents, agents that delay absorption, preservatives. For example, pH adjusting agents include, but are not limited to, phosphate buffer. Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugar, sodium chloride, and the like. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, and the like. Stabilizers have meanings commonly understood by those skilled in the art and can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA (Sucrose-Phosphate-Glutamate-Albumin), sugars (such as Sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as milk white Protein hydrolyzate), etc.

As used herein, examples of mammals include, but are not limited to, humans, non-human primates, rats, mice, cattle, horses, pigs, sheep, alpacas, dogs, cats, and the like. As used herein, the term "subject" refers to a mammal, such as a human. In some embodiments, the subject is human. In some embodiments, the subject is a cancer patient, a human suspected of having or at risk of cancer, or an animal. [ Anti- CD47-CLDN18.2 bispecific antibody ]

The invention provides an anti-CD47-CLDN18.2 bispecific antibody comprising a first antigen-binding portion that binds to CD47 and a second antigen-binding portion that binds to CLDN18.2, wherein the first antigen-binding portion includes a heavy chain variable region (VH) and light chain variable region (VL),

The heavy chain variable region includes: 1) HCDR1, which contains the amino acid sequence of SEQ ID NO: 4 or a variant thereof; 2) HCDR2 comprising the amino acid sequence of SEQ ID NO: 5 or a variant thereof; and 3) HCDR3 comprising the amino acid sequence of SEQ ID NO: 6 or a variant thereof; and

The light chain variable region includes: 1) LCDR1, which contains the amino acid sequence of SEQ ID NO: 7 or a variant thereof; 2) LCDR2 comprising the amino acid sequence of SEQ ID NO: 8 or a variant thereof; and 3) LCDR3, which contains the amino acid sequence of SEQ ID NO: 9 or a variant thereof; Wherein, the variant has a substitution, addition and/or deletion of 1 or 2 amino acids compared to the sequence from which it is derived.

In a specific embodiment, the first antigen binding portion comprises a first antigen binding portion that specifically binds CD47, said first antigen binding portion comprising a heavy chain variable region (VH) and a light chain variable region (VL),

The heavy chain variable region includes: 1) HCDR1, which contains the amino acid sequence of SEQ ID NO: 4; 2) HCDR2, comprising the amino acid sequence of SEQ ID NO: 5; and 3) HCDR3 comprising the amino acid sequence of SEQ ID NO: 6; and

The light chain variable region includes: 1) LCDR1, which contains the amino acid sequence of SEQ ID NO:7; 2) LCDR2, comprising the amino acid sequence of SEQ ID NO: 8; and 3) LCDR3, which contains the amino acid sequence of SEQ ID NO:9.

In one embodiment, the VH comprises: 1) the amino acid sequence of SEQ ID NO: 10; or 2) at least 80%, at least 85%, at least 90%, at least 91%, at least 92% identical to SEQ ID NO: 10 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence identity; and/or

The VL comprises: 1) the amino acid sequence of SEQ ID NO: 11; or 2) having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, Amino acid sequences with at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity.

In one embodiment, the VH comprises the amino acid sequence of SEQ ID NO: 10 and the VL comprises the amino acid sequence of SEQ ID NO: 11.

The first antigen-binding portion may comprise any form of antigen-binding fragment, such as scFv, dsFv, scdsFv, Fab, Fab' or F(ab')2. According to the present invention, the first antigen-binding moiety specifically binds to CD47 on the surface of cancer cells without binding or substantially not binding to CD47 on red blood cells, so that the bispecific antibody of the present invention does not cause red blood cell agglutination.

The second antigen-binding portion may comprise any form of antigen-binding fragment, including, but not limited to, scFv, dsFv, scdsFv, Fab, Fab', F(ab')2, and single variable domains. In some embodiments, the second antigen-binding portion comprises an immunoglobulin single variable domain that specifically binds CLD18.2. Immunoglobulin single variable domains that specifically bind to CLD18.2 are described, for example, in CN112480248A and WO2020238730A1, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the immunoglobulin single variable domain comprises: CDR1, which comprises the amino acid sequence of SEQ ID NO: 13 or a variant thereof; CDR2, which comprises the amino acid sequence of SEQ ID NO: 14 or a variant thereof; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 15 or a variant thereof; wherein the variant has a substitution, addition and/or deletion of 1 or 2 amino acids compared to the sequence from which it is derived. In a specific embodiment, the immunoglobulin single variable domain includes: CDR1, which includes the amino acid sequence of SEQ ID NO: 13; CDR2, which includes the amino acid sequence of SEQ ID NO: 14; and CDR3, which includes Amino acid sequence of SEQ ID NO:15. In one embodiment, the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO: 12. In yet another embodiment, the immunoglobulin single variable domain comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93% of the amino acid sequence of SEQ ID NO: 12. %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

In some embodiments, the first antigen binding moiety and the second antigen binding moiety are linked via a linker. The linker may be a peptide linker or a chemical bond, preferably a peptide linker. Exemplary peptide linkers may include, but are not limited to, polyglycine (G), polyalanine (A), polyserine (S), or combinations thereof, such as GGAS, GGGS, GGGSG, or (G¬4S)n, where n is 1 An integer of -20. Preferably, n is an integer from 1 to 5. In a specific embodiment, the peptide linker comprises the amino acid sequence of SEQ ID NO:22 or SEQ ID NO:23.

In some embodiments, the anti-CD47-CLDN18.2 bispecific antibodies of the invention further comprise an immunoglobulin constant region. The immunoglobulin constant regions can be the heavy chain constant region (CH) and the light chain constant region (CL) of an immunoglobulin of any species. The heavy chain constant region may be derived from any subtype (e.g., IgA, IgD, IgE, IgG, and IgM), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass (e.g., IgG2a and IgG2b) of immunity. The heavy chain constant region of a globulin or a combination thereof. In a preferred embodiment, the heavy chain constant region at least includes an Fc region. For example, the heavy chain constant region of IgG1 may include: all or part of the hinge region-CH2-CH3 or CH1-hinge region-CH2-CH3. The light chain constant region can be derived from a lambda (Lambda) light chain or a kappa (Kappa) light chain constant region. In a preferred embodiment, the heavy chain constant region is that of human IgG1. In one embodiment, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:2. In a preferred embodiment, the light chain constant region is a human kappa light chain constant region. In one embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO:3.

In one embodiment, the VH and VL of the first antigen binding moiety are fused to the N-terminus of the heavy chain constant region and the light chain constant region, respectively, and the single variable domain of the second antigen binding moiety is optionally fused via a linker to The N-terminus of the VH, the N-terminus of the VL, the C-terminus of the heavy chain constant region or the C-terminus of the light chain constant region.

In some embodiments, the anti-CD47-CLDN18.2 bispecific antibody comprises a first polypeptide comprising the VH and heavy chain constant regions of the first antigen-binding portion, and a second polypeptide. The peptide comprises the VL and light chain constant regions of the first antigen binding moiety, the single variable domain of the second antigen binding moiety optionally fused via a linker to the N-terminus of the VH or VL or to the heavy chain constant region or light chain constant region. The C-terminus of the chain constant region. In one embodiment, the single variable domain is optionally fused to the N-terminus of the VH or the C-terminus of the heavy chain constant region through a linker, and the first polypeptide can also be called a fused heavy chain. In one embodiment, the fusion heavy chain has the structure of Formula (I) or Formula (III) as described below. In another embodiment, the single variable domain of the second antigen-binding moiety is optionally fused to the N-terminus of the VL or the C-terminus of the light chain constant region through a linker, and the second polypeptide can also be referred to as Fusion light chain. In one embodiment, the fusion light chain has the structure of Formula (IV) or Formula (VI) as described below.

In one embodiment, the first polypeptide has the structure of formula (I): VH-CH-Linker-VHH Formula (I),

The second polypeptide has the structure of formula (II): VL-CL formula (II),

in

VH and VL are respectively the heavy chain variable region and the light chain variable region of the first antigen-binding portion as described above;

VHH is an immunoglobulin single variable domain as described above;

CH and CL are the heavy chain constant region and light chain constant region respectively as described above;

Linker is a connector.

In one embodiment, the first polypeptide has the structure of formula (I), which includes the amino acid sequence of SEQ ID NO: 20; the second polypeptide has the structure of formula (II), which includes the amino acid sequence of SEQ ID NO: 17 sequence.

In one embodiment, the first polypeptide has the structure of formula (III): VHH-Linker-VH-CH Formula (III),

The second polypeptide has the structure of formula (II),

in

VHH is an immunoglobulin single variable domain as described above;

VH is the heavy chain variable region of the first antigen-binding portion as described above;

CH is the heavy chain constant region as described above;

Formula (II) is as defined above;

Linker is a connector.

In one embodiment, the first polypeptide has the structure of formula (III), which includes the amino acid sequence of SEQ ID NO: 16; the second polypeptide has the structure of formula (II), which includes the amino acid sequence of SEQ ID NO: 17 sequence.

In one embodiment, the first polypeptide has the structure of formula (III), which includes the amino acid sequence of SEQ ID NO: 21; the second polypeptide has the structure of formula (II), which includes the amino acid sequence of SEQ ID NO: 17 sequence.

In yet another embodiment, the first polypeptide has the structure of formula (III) and the second polypeptide has the structure of formula (IV): VHH-Linker-VL-CL Formula (IV),

in

Formula (III) is as defined above;

VHH is an immunoglobulin single variable domain as described above;

VL is the light chain variable region of the first antigen-binding portion as described above;

CL is the light chain constant region as described above;

Linker is a connector.

In one embodiment, the first polypeptide has the structure of formula (III), which includes the amino acid sequence of SEQ ID NO: 16; the second polypeptide has the structure of formula (IV), which includes the amino acid sequence of SEQ ID NO: 19 sequence.

In another embodiment, the first polypeptide has the structure of Formula (V): VH-CH formula (V),

The second polypeptide has the structure of formula (IV),

in

Formula (IV) is as defined above;

VH is the heavy chain variable region of the first antigen-binding portion as described above;

CH is the heavy chain constant region as described above;

Linker is a connector.

In one embodiment, the first polypeptide has the structure of formula (V), which includes the amino acid sequence of SEQ ID NO: 18; the second polypeptide has the structure of formula (IV), which includes the amino acid sequence of SEQ ID NO: 19 sequence.

In another embodiment, the first polypeptide has the structure of Formula (V) and the second polypeptide has the structure of Formula (VI): VL-CL-Linker-VHH Formula (VI),

in

Formula (V) is as defined above;

VHH is an immunoglobulin single variable domain as described above;

VL is the light chain variable region of the first antigen-binding portion as described above;

CL is the light chain constant region as described above;

Linker is a connector.

In some embodiments, the anti-CD47-CLDN18.2 bispecific antibodies of the invention are able to: 1) block the binding of CD47 on the surface of cancer cells to SIRPα; 2) induce macrophages to respond to cancer cells expressing CD47 and CLDN18.2 phagocytosis; and/or 3) bind to cancer cells expressing CD47 and CLDN18.2 without binding or substantially not binding to red blood cells. [ Polynucleotides , vectors and host cells ]

In another aspect, the invention provides an isolated polynucleotide comprising a polynucleotide sequence encoding an anti-CD47-CLDN18.2 bispecific antibody of the invention.

The polynucleotides of the invention can be obtained using methods known in the art, for example, isolated from phage display libraries, yeast display libraries, immunized animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated Immortalized B cells) or chemical synthesis. The polynucleotides of the invention can be codon-optimized for the host cell used for expression.

In yet another aspect, the invention also provides a vector comprising a polynucleotide of the invention. In some embodiments, the polynucleotides of the invention are copied into expression vectors. The expression vector may further contain additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. Expression vectors may also contain polynucleotide sequences encoding additional polypeptides. Additional polypeptides may be, for example, polypeptides that facilitate detection and/or isolation of antibodies or antigen-binding fragments, including but not limited to affinity tags such as polyhistidine tags (His6) or glutathione S-transferase (GST) tags. ), peptides containing protease cleavage sites, and reporter proteins (e.g., fluorescent proteins).

In one embodiment, the polynucleotides of the invention are prepared as recombinant nucleic acids. Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombinant technology (such as polymerase chain reaction (PCR) technology), etc. (see Sambrook, J., E. F. Fritsch, and T. Maniatis. (1989). Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

The polynucleotides of the invention may be present in one or more expression vectors. In some embodiments, the expression vector is a DNA plasmid, such as for expression in bacteria, yeast, or mammalian cells. In other embodiments, the expression vector is a viral vector. In other embodiments, the expression vector is a phage vector or a phagemid vector.

The invention also provides a host cell comprising at least one polynucleotide or vector as described above. Various methods known in the art can be used to introduce the polynucleotide or expression vector of the invention into a suitable host cell. Such methods include, but are not limited to, lipofection, electroporation, viral transduction, calcium phosphate transfection, etc.

In preferred embodiments, host cells are used to express the anti-CD47-CLDN18.2 bispecific antibodies of the invention. Examples of host cells include, but are not limited to, prokaryotic cells (eg bacteria, eg E. coli) and eukaryotic cells (eg yeast, insect cells, mammalian cells).

Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, HeLa cells, HEK cells (eg, HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for antibody expression.

The invention also provides a method for producing the anti-CD47-CLDN18.2 bispecific antibody of the invention, which includes the following steps:

(I) culturing the host cells of the present invention under appropriate conditions to express anti-CD47-CLDN18.2 bispecific antibodies, and

(II) Isolating said antibody from host cells or cultures thereof.

In some embodiments, a single vector is used that contains polynucleotide sequences encoding heavy and light chains. In some embodiments, two vectors are used, one encoding the light chain of the antibody and the other encoding the heavy chain of the antibody. In some embodiments, the host cell also contains a partner plasmid, which can help increase the solubility, stability, and/or folding of the antibody. Techniques for isolating and purifying antibodies from host cells or their culture media are well known to those skilled in the art. [ Antibody conjugate ]

The invention also provides an antibody conjugate comprising an anti-CD47-CLDN18.2 bispecific antibody of the invention conjugated to at least one therapeutic agent. Antibody-drug conjugates (ADCs) are a typical antibody conjugate, where the therapeutic agent can be, for example, a cytotoxic agent.

As used herein, "conjugated" means that two or more moieties are connected to each other through covalent or non-covalent interactions. In preferred embodiments, the conjugation is covalent conjugation.

The therapeutic agent may be selected from the group consisting of cytotoxic agents, therapeutic antibodies (e.g., antibodies that specifically bind to another antigen or antigen-binding fragments thereof), radioisotopes, oligonucleotides and their analogs (e.g., interfering RNA), biologically active Peptides, protein toxins (e.g. diphtheria toxin, ricin) and enzymes (e.g. urease).

Cytotoxic agents refer to substances that inhibit or reduce the activity, function and/or kill cells. Examples of cytotoxic agents may include, but are not limited to: maytansinoid (e.g., maytansine), aristatins (e.g., MMAF, MMAE, MMAD), duostatin, nodida Cyclic peptides (cryptophycin), vinca alkaloids (such as vinblastine, vincristine), colchicines, Aplysia toxins, taxanes, paclitaxel, docetaxel, cabazitaxel, enedynes Antibiotics, cytochalasins, camptothecins, anthracyclines (e.g. daunorubicin, dihydroxyanthracindione, doxorubicin), cytotoxic antibiotics (e.g. silk Mitomycin, actinomycin, duocarmycin (such as CC-1065), auromycin, duomycin, calicheamicin, Endomycin, phenomycin, doxorubicin, daunorubicin, calicheamicin, cisplatin, ethidiumbromide, bleomycin, silk Schizothromycin, radimycin, pradilactone, podophyllotoxin, etoposide, mitoxantrone, 5-fluorouracil, cytarabine, gemcitabine, mercaptopurine, pentostatin, fluoride Darabine, cladribine, nelarabine, carmustine, lomustine, methotrexate, phenylalanine mustard, tenoposide, glucocorticoids, etc.

The radioactive isotope may be selected from, for example, 212Bi, 213Bi, 131I, 125I, 111In, 177Lu, 186Re, 188Re, 153Sm, 90Y. Antibodies labeled with radioactive isotopes are also called radioimmunoconjugates.

In preferred embodiments, the biologically active polypeptide is a polypeptide or protein with therapeutic, binding or enzymatic activity. Non-limiting examples of biologically active polypeptides may include, but are not limited to: protein toxins (eg, diphtheria toxin, ricin), enzymes (eg, urease, horseradish peroxidase), cytokines.

In one embodiment, the therapeutic agent is a molecule with anti-tumor biological activity. Molecules with anti-tumor biological activity include, but are not limited to, cytotoxic agents, chemotherapeutic agents, radioactive isotopes, immune checkpoint inhibitors, antibodies targeting tumor-specific antigens, and other anti-tumor drugs. In a preferred embodiment, the therapeutic agent is a cytotoxic agent. In yet another preferred embodiment, the therapeutic agent is a radioactive isotope.

Therapeutic agents can be conjugated to the anti-CD47-CLDN18.2 bispecific antibodies of the invention through a linker using any technique known in the art. The linker may contain reactive groups for covalent conjugation, such as amine, hydroxylamine, maleimide, carboxyl, phenyl, thiol, sulfhydryl or hydroxyl groups. Joints may be cuttable or non-cuttable. Cleavable linkers include enzyme-cleavable linkers (eg, peptides containing protease cleavage sites), pH-sensitive linkers (eg, hydrazone linkers), or reducible linkers (eg, disulfide bonds).

In one embodiment, the linker contains a reactive group selected from the group consisting of amine, hydroxylamine, maleimide, carboxyl, phenyl, thiol, sulfhydryl and hydroxyl. In one embodiment, the linker is a chemical bond. In one embodiment, the linker comprises an amino acid or a peptide consisting of 2-10 amino acids. Amino acids can be natural or unnatural amino acids.

In some embodiments, the therapeutic agent and linker are conjugated to form an intermediate prior to conjugation to the anti-CD47-CLDN18.2 bispecific antibodies of the invention. In some embodiments, the intermediate is linked by forming a thioether bond with the sulfhydryl group of the anti-CD47-CLDN18.2 bispecific antibody of the invention. The structures and preparation methods of such intermediates and methods of using them to prepare antibody conjugates are described, for example, in International Patent Application Publication No. WO2019114666, the relevant content of which is incorporated herein by reference in its entirety. [ Pharmaceutical composition ]

The present invention also provides pharmaceutical compositions comprising the anti-CD47-CLDN18.2 bispecific antibody or antibody conjugate of the present invention, and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers may include, but are not limited to: diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, excipients, etc. Excipients, preservatives, antioxidants (such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, Propyl gallate, alpha-tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.), solubilizer, gelling agent, softener, solvent (e.g., water, Alcohols, acetic acid, and syrups), buffers (e.g., phosphate buffers, histidine buffers, and acetate buffers), surfactants (e.g., nonionic surfactants such as polysorbate 80, polysorbate 20 , poloxamer or polyethylene glycol), antibacterial agents, antifungal agents, isotonic agents (e.g. trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption delaying agents, chelating agents and emulsifiers . For compositions comprising antibodies or antibody conjugates, suitable carriers may be selected from buffers (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine buffer salt buffer), isotonic agents (such as trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactants (such as polysorbate 80, polysorbate 20, poloxamer) or other combination.

Pharmaceutical compositions provided herein may be in a variety of dosage forms, including, but not limited to, solid, semi-solid, liquid, powder, or lyophilized forms. Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg by injection or infusion). For compositions containing antibodies or antibody conjugates, preferred dosage forms may generally be, for example, injection solutions and lyophilized powders.

The pharmaceutical compositions provided herein may be administered to a subject by any method known in the art, such as by systemic or topical administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavity), topical (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal , rectal, sublingual or local). It will be understood by those skilled in the art that the exact dosage administered will depend on various factors such as the metabolic kinetic properties of the pharmaceutical composition, the duration of treatment, the rate of excretion of the particular compound, the purpose of treatment, the route of administration and the subject's Conditions, such as the patient's age, health, weight, gender, diet, medical history, and other factors known in the medical field. The method of administration may be, for example, injection or infusion.

As a general guide, the anti-CD47-CLDN18.2 bispecific antibodies of the invention may be administered in a dosage range of about 0.0001 to 100 mg/kg, more typically 0.01 to 20 mg/kg of subject body weight. For example, the dosage can be 0.3 mg/kg body weight, 1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight or 20 mg/kg body weight, or between 1-20 mg/kg body weight. within the range of kg. Exemplary treatment regimens call for weekly dosing, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, once every 3-6 months, or with initial dosing intervals slightly The dosing interval is lengthened in the short term. The method of administration can be intravenous drip. [ treatment ]

In yet another aspect, the invention relates to the use of an anti-CD47-CLDN18.2 bispecific antibody, antibody conjugate or pharmaceutical composition of the invention for the manufacture of a medicament for treating a disease in a subject.

The invention also relates to the anti-CD47-CLDN18.2 bispecific antibody, antibody conjugate or pharmaceutical composition of the invention for use in the treatment of diseases.

The invention also provides a method of treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-CD47-CLDN18.2 bispecific antibody, antibody conjugate or medicament of the invention composition.

In one embodiment, the disease as described above is cancer. As used herein, "cancer" includes, but is not limited to, blood cancers and solid tumors. The cancer can also be metastatic. Metastasis is when cancer cells spread from their original site to other parts of the body. For example, anti-CD47-CLDN18.2 bispecific antibodies can be used to treat CLDN18.2-positive cancers. In preferred embodiments, anti-CD47-CLDN18.2 bispecific antibodies are used to treat CD47 and CLDN18.2 positive cancers. In some embodiments, the cancer is gastric cancer. [ Kit ]

The invention also provides a kit comprising the anti-CD47-CLDN18.2 bispecific antibody, antibody conjugate or pharmaceutical composition of the invention, and instructions for use. The kit may also contain suitable containers. In certain embodiments, the kit further includes a device for administering the drug. Typically, the kit also includes a label indicating the intended use and/or method of use of the contents of the kit. The term "label" includes any written or recorded material on or provided with the kit or otherwise provided with the kit. [ beneficial effect ]

The anti-CD47-CLDN18.2 bispecific antibody of the present invention can achieve at least one of the following beneficial effects:

1) Block CD47 on the surface of cancer cells from binding to SIRPα;

2) Inducing macrophages to phagocytose cancer cells expressing CD47 and CLDN18.2;

3) mediate ADCP against CLDN18.2-expressing cancer cells; and

4) Binds to cancer cells expressing CD47 and CLDN18.2 without binding or basically not binding to red blood cells.

In addition, compared with the monoclonal antibody that targets CD47 alone, the anti-CD47-CLDN18.2 bispecific antibody of the present invention has higher binding activity to CD47 and CLDN18.2-positive tumor cells, and has a higher binding activity to CD47 and SIRPα. The blocking effect is stronger. [ Example ]

The following examples are intended to illustrate the invention only and therefore should not be construed as limiting the invention in any way. [ Materials and methods ]

1. Construction of antibody expression plasmids

DNA fragments encoding the antibody heavy chain and light chain were prepared through DNA recombinant technology, and then copied to the expression vector pcDNA3.4-TOPO (Invitrogen) to obtain the antibody heavy chain and light chain expression plasmids. Antibody heavy and light chain expression plasmids were amplified and subsequently extracted and purified in E. coli DH5α.

2. Antibody expression and purification

All antibodies were expressed through the ExpiCHO transient expression system (Thermo Fisher, A29133) (see WO2020238730A1) and affinity purified using MabSelect SuRe LX (GE, 17547403).

3. SDS-PAGE Identify antibody purity

Preparation of reducing solution: Add 2 µg of the antibody to be tested or IPI (Ipilimlumab; control) into 5×SDS loading buffer (containing DTT with a final concentration of 5 mM), heat in a dry bath at 100 °C for 10 min, and cool to room temperature. After warming, centrifuge at 12,000 rpm for 5 min and take the supernatant. The supernatant was added to Bis-tris 4-15% gradient gel (Genscript) for protein gel electrophoresis. The protein bands were then stained with Coomassie brilliant blue, destained and scanned with an EPSON V550 color scanner, and the purity of the protein bands was calculated using ImageJ according to the peak area normalization method.

4. SEC-HPLC Identify antibody purity

The Agilent HPLC 1100 column (XBridge BEH SEC 3.5µm, 7.8 mm I.D.×30 cm, Waters) flow rate was set to 0.8 mL/min, the injection volume was 20 µL, and the VWD detector wavelength was 280 nm and 214 nm. The mobile phase used 150 mmol/L phosphate buffer, pH 7.4. All samples were diluted to 0.5 mg/mL using the mobile phase, and then the blank solution and sample solution were injected sequentially. Calculate the percentage of high molecular aggregates, antibody monomers and low molecular aggregates in the sample according to the area normalization method.

5. Differential scanning Fluorescence method to identify antibody stability

Differential scanning fluorescence (DSF) can provide information about the structural stability of a protein based on the fluorescence change process in the protein map, detect the conformational changes of the protein, and obtain the melting temperature (Tm) of the protein.

Prepare a 0.2 mg/mL antibody sample solution to be tested, and use PBS and IPI (Ipilimlumab; 0.2 mg/mL) as controls. The test samples were added to a 96-well plate (Nunc) in triplicate at 19 μL/well, and then 1 μL of 100×SYPRO orange dye was added to each well, piped and mixed with a microdispenser, and prepared for use on the machine. ABI 7500 FAST RT-PCR instrument was used to test the thermal stability of the sample. Melting curve was selected as the test type and continuous mode was used. The scanning temperature range was 25~95°C, the heating rate was 1%, and the temperature was equilibrated at 25°C for 5 minutes. Data were collected during the heating process. , select "ROX" as the reporter group, select "None" as the quencher group, the reaction volume is 20 μL, and the temperature corresponding to the first peak and valley of the first derivative of the melting curve is determined as the melting temperature Tm of the antibody. [ Example 1 - Design of anti -CD47-CLDN18.2 bispecific antibody ]

This example describes an exemplary anti-CD47-CLDN18.2 bispecific antibody in which the CLDN18.2-binding arm employs an anti-CLDN18.2 single domain antibody NA3S that specifically recognizes human CLDN18.2 but not CLDN18.1 -H1 (the amino acid sequences of CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 13, 14 and 15 respectively; the amino acid sequence of VHH is shown in SEQ ID NO: 12), the CD47-binding arm uses the anti-CD47 humanized antibody A7H3L3 The amino acid sequences of the antigen-binding domains (HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO: 4, 5 and 6 respectively; the amino acid sequences of LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 7, 8 and 9 respectively; the heavy chain can The amino acid sequence of the variable region is shown in SEQ ID NO: 10, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 11). The heavy chain variable region of antibody A7H3L3 was fused to the human IgG1 heavy chain constant region (SEQ ID NO: 2) to form the heavy chain of antibody A7H3L3, and the light chain variable region was fused to the human kappa light chain constant region (SEQ ID NO: 3 ) forms the light chain of antibody A7H3L3. The anti-CLDN18.2 single domain antibody NA3S-H1 has been published in WO2020238730A1. Its in vitro ADCC and CDC cell killing effects and tumor inhibition tests on the human CLDN18.2-HEK29T-SCID tumor transplant model have shown excellent drug efficacy. effect. The humanized antibody A7H3L3 binds very weakly to CD47 on red blood cells and is an ideal candidate for bispecific antibodies. At the same time, the design of the bispecific antibody will make the binding arm of the anti-CD47 antibody A7H3L3 better because it binds CLDN18.2. Combined with tumor cells expressing dual targets, it can better block SIRPα inhibitory signals at the same time.

Bispecific antibodies were designed based on the valence, position and linker length of the anti-CLDN18.2 single domain antibody (VHH) NA3S-H1, and 5 bispecific antibodies (B8-B12 respectively) were designed. Exemplary bispecific antibody structures are shown in Table 1 and Figure 1, and the corresponding amino acid sequences are provided in Table 2, where the sequence of Linker1 is GGGGSGGGS (SEQ ID NO: 22), and the sequence of Linker2 is GGGGS (SEQ ID NO: twenty three). Table 1: Anti-CD47-CLDN18.2 bispecific antibody structure Antibody name Heavy chain structure light chain structure B8 VHH ^CLDN18.2 -Linker1-VH ^CD47 -CH1-Hinge-CH2-CH3 VL ^CD47 -CL B9 VH ^CD47 -CH1-Hinge-CH2-CH3 VHH ^CLDN18.2 -Linker1-VL ^CD47 -CL B10 VH ^CD47 -CH1-Hinge-CH2-CH3-Linker1- VHH ^CLDN18.2 VL ^CD47 -CL B11 VHH ^CLDN18.2 -Linker1-VH ^CD47 -CH1-Hinge-CH2-CH3 VHH ^CLDN18.2 -Linker1-VL ^CD47 -CL B12 VHH ^CLDN18.2 -Linker2-VH ^CD47 -CH1-Hinge-CH2-CH3 VL ^CD47 -CL Table 2: Anti-CD47-CLDN18.2 bispecific antibody amino acid sequence Antibody name heavy chain amino acid sequence light chain amino acid sequence B8 SEQ ID NO:16 SEQ ID NO:17 B9 SEQ ID NO:18 SEQ ID NO:19 B10 SEQ ID NO:20 SEQ ID NO:17 B11 SEQ ID NO:16 SEQ ID NO:19 B12 SEQ ID NO:21 SEQ ID NO:17 [ Example 2 - Binding activity of anti -CD47-CLDN18.2 bispecific antibody to CD47 on red blood cells ]

Flow cytometry was used to determine whether the bispecific antibody bound to red blood cells. Another anti-CD47 antibody F4AM4-IgG1 developed by the applicant (abbreviated as F4AM4 in the drawings; the amino acid sequence of the heavy chain is SEQ ID NO: 24, and the amino acid sequence of the light chain is SEQ ID NO: 25) was used as a positive control. antibody. F4AM4-IgG1 showed strong binding to CD47 of red blood cells in a series of functional verification experiments (conducted before the experiments of this example) and was therefore selected as a positive control in this example.

The specific method is as follows: Separate red blood cells from 1 mL of anticoagulated human blood, centrifuge and aspirate the supernatant, rinse twice with PBS, add 1 mL of PBS and resuspend. Use PBS to dilute the red blood cells to 1×10 ⁷ /mL, add 50 μL of red blood cells into each well into a 96-well round-bottom cell culture plate, then add equal volumes of serially diluted antibodies to be tested, mix thoroughly, and place at 4°C Incubate for 1 h. Then rinse with FACS buffer three times, add 0.5 μg of PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596), and incubate at 4°C for 1 h. Afterwards, the cells were washed three times with FACS buffer, and 200 μL of FACS buffer was added to the cells to resuspend the cells. Finally, the amount of antibodies bound to red blood cells was detected by flow cytometry (Beckman, CytoFLEX AOO-1-1102) ( Expressed as mean fluorescence intensity (MFI)).

The binding activity of the antibodies to red blood cells is shown in Figures 2a and 2b. As shown in Figures 2a and 2b, even at very high concentrations (150 μg/mL), all anti-CD47-CLDN18.2 bispecific antibodies barely bound to red blood cells relative to the strong binding of F4AM4-IgG1 on red blood cells. Or the binding activity is extremely low; at high concentration (15 μg/mL), there is no significant difference in the binding of B9, B10 and B11 to the negative control IgG1 on red blood cells. The specific values are shown in Table 3. It can be seen that the bispecific antibody of the present invention has low binding activity to CD47 on red blood cells and basically does not cause red blood cell agglutination. Table 3: Mean fluorescence intensity (MFI) of bispecific antibody binding on red blood cells B8 B9 B10 B11 B12 A7H3L3 IgG1 F4AM4-IgG1 15 μg/mL MFI 988 239 534 238 1615 463 303 63000 150 μg/mL MFI 2145 1220 2493 847 2526 608 901 ＞63000 [ Example 3 - Binding activity of anti -CD47-CLDN18.2 bispecific antibody on tumor cells expressing single and dual targets ]

The effect of anti-CD47-CLDN18.2 bispecific antibody B10 on NUGC-4 cells (expressing endogenous CD47, purchased from BNCC strain library, No. BNCC341962) and hCLDN18.2-NUGC-4 cells (using Lentiviral transfection was used to construct the gastric cancer cell line NUGC-4 that overexpresses exogenous human CLDN18.2 (amino acid sequence SEQ ID NO: 1) and simultaneously expresses the binding activity of endogenous CD47). For comparison, the binding activities of antibodies 1F8 (antibody 1F8 in WO2018075857A1) and F4AM4-IgG1 against these two cell lines were also determined. Human IgG1 was used as isotype negative control.

The specific method is as follows: take 1×105 NUGC-4 cells or hCLDN18.2-NUGC-4 cells, centrifuge at low speed (300 g) to remove the supernatant; pass the cells at the bottom of the centrifuge tube through the prepared FACS buffer (including volume percentage). Rinse once with 2% FBS (1×PBS buffer), then add gradient dilutions of the antibody to be tested to the washed cells, and incubate at 4°C for 1 h; then rinse three times with the above FACS buffer, add PE-labeled goat anti-human IgG Fc antibody (Abcam, ab98596) 0.5 μg, incubated at 4°C for 1 h; then the cells were washed three times with FACS buffer and resuspended in 200 μL FACS buffer, and finally passed through the flow cytometer (Beckman, CytoFLEX AOO-1-1102) detects the amount of antibody bound to red blood cells (expressed as mean fluorescence intensity (MFI)).

The binding activities of the antibodies to NUGC-4 cells and hCLDN18.2-NUGC-4 cells are shown in Figures 3a and 3b, respectively. As shown in Figure 3a, the binding activity of bispecific antibody B10 on tumor cells NUGC-4 was weak and slightly weaker than antibody 1F8. As shown in Figure 3b, bispecific antibody B10 has significantly better binding activity than antibody 1F8 on hCLDN18.2-NUGC-4 cells expressing both CD47 and CLDN18.2. Based on this, although the bispecific antibody B10 binds weaker than 1F8 to cells expressing CD47 single target, it binds better than 1F8 to cells expressing both CD47 and CLDN18.2. This result proves that the bispecific antibody B10 can simultaneously bind to CLDN18.2 and CD47 in tumor cells, increasing the ability to bind to tumor cells. [ Example 4 - The ability of anti -CD47-CLDN18.2 bispecific antibody to block the binding of CD47 to SIRPα on tumor cells expressing single and dual targets ]

The ability of the anti-CD47-CLDN18.2 bispecific antibody to block the binding of CD47 to SIRPα on NUGC-4 and hCLDN18.2-NUGC-4 cells was determined by flow cytometry. For comparison, the ability of antibodies 1F8, F4AM4-IgG1, and A7H3L3 to block the binding of CD47 to SIRPα on NUGC-4 and hCLDN18.2-NUGC-4 tumor cells was also determined.

The specific method is as follows: take 1×10 ⁵ NUGC-4 cells or hCLDN18.2-NUGC-4 cells, centrifuge at low speed (300 g) and remove the supernatant. Rinse the cells at the bottom of the centrifuge tube once with the prepared FACS buffer (1×PBS buffer containing 2% FBS); then add gradient dilutions of the antibody to be tested to the washed cells, and incubate for 1 hour; Rinse the cells twice with FACS buffer, add 100 μL of 1 μg/mL SIRPα-mFc (ACRO, SIA-H52A8), and incubate for 1 h at 4°C; rinse the cells three times with FACS buffer, add 100 μL of 1:200 dilution PE-labeled goat anti-mouse Fc secondary antibody (Abcam, ab98742), incubated at 4°C for 1 h, centrifuged to remove the supernatant, added 200 μL FACS buffer to the cells to resuspend the cells, and finally passed through the flow cytometer (Beckman, CytoFLEX AOO-1-1102) detects the amount of SIRPα-mFc bound to cells (expressed as mean fluorescence intensity (MFI)).

The results on NUGC-4 cells are shown in Figure 4a: Antibody F4AM4-IgG1 can effectively block the binding of CD47 to SIRPα, with an IC ₅₀ of 0.033 μg/mL (0.226 nM); antibody 1F8 has a weak blocking effect. , IC ₅₀ is 15.36 μg/mL (105.6 nM); while bispecific antibody B10 has almost no blocking ability.

The results on hCLDN18.2-NUGC-4 cells are shown in Figure 4b: Antibody F4AM4-IgG1 still has strong blocking ability on this tumor cell, with an IC ₅₀ of 0.056 μg/mL (0.383 nM); compared to In terms of blocking ability on NUGC-4 cells, the blocking ability of bispecific antibody B10 on hCLDN18.2-NUGC-4 cells is significantly improved and is better than antibody 1F8; B10 and 1F8 block hCLDN18.2-NUGC- The IC ₅₀ of CD47 binding to SIRPα on 4 are 0.765 μg/mL (4.476 nM) and 11.98 μg/mL (82.34 nM) respectively; in addition, the anti-CLDN18.2 single domain antibody NA3S-H1 only binds to CLDN18.2, so there is no Any blocking effect. Based on this, although the blocking activity of bispecific antibody B10 on cells expressing CD47 single target is weaker than that of antibody 1F8, its blocking activity on cells expressing dual targets of CD47 and CLDN18.2 is significantly better than antibody 1F8. It will exert a stronger ability to block the binding of CD47 to the receptor SIRPα.

The blocking ability of other bispecific antibodies on hCLDN18.2-NUGC-4 cells was also determined, and the results are shown in Figure 4c. Among bispecific antibodies B8-B12, bispecific antibody B10 has the strongest blocking activity. [ Example 5 - In vivo tumor inhibition experiment of anti -CD47-CLDN18.2 bispecific antibody ]

5.1 In vivo tumor suppression experiment 1

Female nude mice (16-18 g) aged 6-7 weeks were kept in independent ventilated boxes with constant temperature and humidity. The temperature of the breeding room was 21-24°C and the humidity was 30-53%. 3×106 hCLDN18.2-NUGC-4 cells were subcutaneously injected into the left axilla of nude mice (day 0). When the subcutaneous tumor-bearing volume of the mice reached about 300-400 ^mm3 (day 20), the tumors were removed. Mouse samples with large volume differences were then randomly divided into groups according to tumor volume (8 mice per group): PBS treatment group, NA3S-H1 monoclonal antibody administration group, A7H3L3 monoclonal antibody administration group, NA3S-H1+ A7H3L3 combined administration group and bispecific antibody B10 administration group. Taking NA3S-H1 monoclonal antibody 5 mg/kg as the standard, all other drugs are administered in equimolar doses, that is, A7H3L3 monoclonal antibody 9.4 mg/kg, NA3S-H1+ A7H3L3 combined with 5 mg/kg+9.4 mg/kg. , bispecific antibody B10 10.6 mg/kg. It is administered twice a week, alternating between intraperitoneal injection (ip) and intravenous injection (iv). Observe and record the tumor length (mm) and width (mm) at any time, and calculate the tumor volume (V). The calculation method is: V = (length × width 2)/2, tumor inhibition rate TGI (%) = (1-give Average tumor volume of the drug group/average tumor volume of the PBS-treated group) × 100%.

The results of antibody tumor inhibition are shown in Figure 5a and Table 4. It can be seen from this that: at this equimolar dose, the NA3S-H1 monoclonal antibody administration group showed almost no tumor inhibition effect, and the other groups all showed certain tumor inhibition effects. Among them, the bispecific antibody B10 has the best inhibitory effect, reaching a tumor inhibition rate of nearly 54.54% before the 39th day. The tumor size is close to the initial tumor volume on the 20th day, and is better than the combined drug (A7H3L3+NA3S-H1 ( 9.4+5 mpk)), which indicates that the binding of bispecific antibody B10 to CLDN18.2 can enhance its blocking effect on the binding of CD47 and SIRPα. Table 4: Tumor inhibition rate of bispecific antibodies in mice days NA3S-H1 A7H3L3 B10 A7H3L3+NA3S-H1 25 2.96% -1.31% 3.41% 12.73% 28 8.76% 2.17% 27.69% 18.40% 32 -5.33% 3.73% 40.26% 19.30% 35 -6.94% 2.10% 44.31% 19.49% 39 2.01% 15.00% 54.54% 33.71% 42 -7.26% 14.65% 51.45% 30.84% 46 -8.69% 24.65% 48.33% 32.59% 49 -5.94% 25.18% 45.12% 25.62%

5.2 In vivo tumor suppression experiment 2

Randomly grouped (8 mice per group): PBS treatment group, monoclonal antibody NA3S-H1 administration group, and bispecific antibody B10 administration group. The first dose was given on the day of vaccination (day 0), and was given twice a week. NA3S-H1 monoclonal antibody 2.5 mg/kg was used as the standard, and the bispecific antibody B10 was given in an equimolar dose, that is, 5.3 mg. /kg. The rest is consistent with Example 5.1.

The results are shown in Figure 5b. At this equimolar dose, the NA3S-H1 monoclonal antibody administration group showed a certain tumor inhibitory effect, with a tumor inhibition rate of approximately 54.99% (day 34); all bispecific antibody B10 groups Tumors in mice were completely suppressed, and the tumor inhibition rate was close to 100% (day 34). This result proves that the tumor inhibitory effect of bispecific antibody B10 is significantly better than that of anti-CLDN18.2 monoclonal antibody NA3S-H1. [ Example 6 - Determination of physical and chemical properties of anti -CD47-CLDN18.2 bispecific antibody ]

6.1 SDS-PAGE Purity identification

Reducing SDS-PAGE was used to identify the purity of bispecific antibody B10. The apparent relative molecular weights of the heavy chain and light chain main bands of bispecific antibody B10 are about 65 kD and 25 kD respectively, which is in line with the expected size, and the purity is about 90%.

6.2 SEC-HPLC Purity identification

SEC-HPLC was used to detect the monomer purity of bispecific antibody B10. The monomer purity of bispecific antibody B10 measured by SEC-HPLC was greater than 94%.

6.3 bispecific antibodies B10 of DSF Thermal stability testing

The Tm value of bispecific antibody B10 was detected using DSF method to evaluate its thermal stability. The results show that bispecific antibody B10 has two melting peaks, the first peak Tm value is 69.85 ± 0.06 ℃, and the second peak Tm value is 80.60 ± 0.16 ℃, indicating that bispecific antibody B10 has good thermal stability . [ Example 7 - ADCP activity of anti -CD47-CLDN18.2 bispecific antibody ]

Antibody-dependent cell-mediated phagocytosis (ADCP) is an important mechanism of action of therapeutic antibodies against viral infections or tumor cells. This experiment evaluates the ADCP activity of the bispecific antibody of the present invention through a bioluminescent reporter gene method. This method uses genetically engineered Jurkat cells (BPS Bioscience Inc., 71273) as effector cells, which stably express FcγRⅡa receptors and express luciferase driven by NFAT response elements (Int Immunopharmacol. 2021 Nov;100:108- 112.). After the antibody recognizes the target cell, it binds to FcγRⅡa on the surface of the effector cell and stimulates the NFAT response element in the cell. The NFAT response element drives the expression of luciferase. Luciferase activity can be quantified by bioluminescence.

Dilute the samples to be tested (B10, A7H3L3, NA3S-H1) to the initial reaction concentration of 200 nM, and dilute 10 gradients at 2 times. Add the diluted sample to a 96-well white bottom plate, 50 μL per well, and set 3 replicates for each concentration gradient. Add 150 μL/well of PBS solution to the edge wells of the 96-well white bottom plate. Then, 5 105 cells/mL of hCLDN18.2-NUGC-4 cells (target cells) were added to the 96-well white bottom plate, 50 μL/well, and incubated at room temperature for 30 min. Then add 1.5 106 effector cells/mL as described above, 50 μL/well. The 96-well white bottom plate was cultured in a 37°C 5% CO2 incubator for 6 h. Detection: Take out the 96-well white bottom plate and let it stand at room temperature for 30 minutes. Add the detection reagent luciferase substrate (Bio-Glo™ Luciferase Assay System Promega G7940), 50 μL/well, react at room temperature in the dark for 5-10 minutes, and detect the luminescence intensity through a microplate reader (expressed as relative light units ( RLU)). The logarithm of the antibody concentration was plotted against the RLU value at the corresponding concentration, the data were analyzed using Graphpad Prism software, and the EC ₅₀ value was calculated.

Experimental results: It can be seen from Figure 6 and Table 5 that when the target cells are hCLDN18.2-NUGC-4 cells, compared with the anti-CD47 monoclonal antibody A7H3L3 and the anti-CLDN18.2 monoclonal antibody NA3S-H1, the dual specificity of the present invention Antibody B10 showed higher ADCP activity. Table 5: ADCP activity of bispecific antibodies NA3S-H1 B10 A7H3L3 EC ₅₀ 7.169 11.69 25.64 R ² 0.9948 0.9895 0.9962

Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings disclosed herein, and these changes are within the protection scope of the present invention. The scope of protection of the present invention is given by the appended patent claims and any equivalents thereof.

without

Figure 1 shows the schematic structure of the anti-CD47-CLDN18.2 bispecific antibody; Figure 2a and Figure 2b show the binding activity of anti-CD47-CLDN18.2 bispecific antibody to CD47 on red blood cells; Figure 3a and Figure 3b show the binding activity of the anti-CD47-CLDN18.2 bispecific antibody on tumor cells expressing single and dual targets. Figure 3a shows the binding activity of the anti-CD47-CLDN18.2 bispecific antibody on NUGC. Binding activity on -4 cells, Figure 3b shows the binding activity of anti-CD47-CLDN18.2 bispecific antibody on hCLDN18.2-NUGC-4 cells; Figure 4a, Figure 4b and Figure 4c show the ability of the anti-CD47-CLDN18.2 bispecific antibody to block the binding of human CD47 to the receptor SIRPα on tumor cells expressing single and dual targets, wherein Figure 4a shows The ability of the anti-CD47-CLDN18.2 bispecific antibody to block the binding of human CD47 to the receptor SIRPα on NUGC-4 cells. Figure 4b and Figure 4c show that the anti-CD47-CLDN18.2 bispecific antibody blocks the binding of human CD47 to the receptor SIRPα on hCLDN18.2- The ability to block the binding of human CD47 to the receptor SIRPα on NUGC-4 cells; Figures 5a and 5b show the inhibitory effect of anti-CD47-CLDN18.2 bispecific antibody on tumor growth in mice; and Figure 6 shows the ADCP activity of anti-CD47-CLDN18.2 bispecific antibody measured by bioluminescence reporter gene method.

TW202321297A_111139359_SEQL.xml

Claims (12)

A bispecific antibody comprising a first antigen-binding portion that binds CD47 and a second antigen-binding portion that binds CLDN18.2, wherein the first antigen-binding portion includes a heavy chain variable region (VH) and a light chain variable region (VH). chain variable region (VL), The heavy chain variable region includes: 1) HCDR1, which contains the amino acid sequence of SEQ ID NO:4; 2) HCDR2, comprising the amino acid sequence of SEQ ID NO: 5; and 3) HCDR3 comprising the amino acid sequence of SEQ ID NO: 6; and The light chain variable region includes: 1) LCDR1, which contains the amino acid sequence of SEQ ID NO:7; 2) LCDR2, comprising the amino acid sequence of SEQ ID NO: 8; and 3) LCDR3, which contains the amino acid sequence of SEQ ID NO:9. The bispecific antibody as described in claim 1, wherein The heavy chain variable region includes: 1) the amino acid sequence of SEQ ID NO: 10; or 2) At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least Amino acid sequences with 98% or at least 99% sequence identity; and/or The light chain variable region includes: 1) The amino acid sequence of SEQ ID NO: 11; or 2) At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least SEQ ID NO: 11 Amino acid sequences with 98% or at least 99% sequence identity; Preferably, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 10, and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 11. The bispecific antibody of claim 1 or 2, wherein the second antigen-binding portion comprises an immunoglobulin single variable domain (VHH) that binds CLDN18.2; Preferably, the immunoglobulin single variable domain comprises: CDR1, which includes the amino acid sequence of SEQ ID NO: 13; CDR2 comprising the amino acid sequence of SEQ ID NO: 14; and CDR3, which includes the amino acid sequence of SEQ ID NO: 15; More preferably, the immunoglobulin single variable domain comprises: 1) The amino acid sequence of SEQ ID NO: 12; or 2) At least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% with the amino acid sequence of SEQ ID NO: 12 %, at least 98% or at least 99% sequence identity of the amino acid sequence. The bispecific antibody according to any one of claims 1 to 3, wherein the first antigen-binding portion and the second antigen-binding portion are connected through a linker. The bispecific antibody according to any one of claims 1 to 4, further comprising a heavy chain constant region (CH) and a light chain constant region (CL) of an immunoglobulin; Preferably, the heavy chain constant region is that of human IgG1, and/or The light chain constant region is a human kappa light chain constant region; More preferably, the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:2; and/or the light chain constant region comprises the amino acid sequence of SEQ ID NO:3. The bispecific antibody of claim 5, which includes a first polypeptide and a second polypeptide, in The first polypeptide has the following structure from N-terminus to C-terminus: VH-CH-Linker-VHH, The second polypeptide has the following structure from N-terminus to C-terminus: VL-CL, in VH and VL are the heavy chain variable region and the light chain variable region as defined in claim 1 or 2 respectively; VHH is an immunoglobulin single variable domain as defined in claim 3; CH and CL are the heavy chain constant region and the light chain constant region respectively as defined in claim 5; Linker is the connector; Preferably, the first polypeptide comprises the amino acid sequence of SEQ ID NO:20 and the second polypeptide comprises the amino acid sequence of SEQ ID NO:17. An isolated polynucleotide encoding the bispecific antibody of any one of claims 1-6. An expression vector comprising the polynucleotide described in claim 7. A host cell comprising the polynucleotide according to claim 7 or the expression vector according to claim 8. An antibody conjugate comprising the bispecific antibody of any one of claims 1-6 conjugated to at least one therapeutic agent. A pharmaceutical composition comprising the bispecific antibody as described in any one of claims 1-6 or the antibody conjugate as described in claim 10, and a pharmaceutically acceptable carrier. A bispecific antibody as described in any one of claims 1-6, an antibody conjugate as described in claim 10, or a pharmaceutical composition as described in claim 11 in the preparation of medicines for treating cancer Use; Preferably, the cancer is gastric cancer.

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